Isolation, identification, degradation mechanism and exploration of active enzymes in the ochratoxin A degrading strain Acinetobacter pittii AP19.

Ochratoxin A (OTA) is a polyketide mycotoxin that commonly contaminates agricultural products and causes significant economic losses. In this study, the efficient OTA-degrading strain AP19 was isolated from vineyard soil and was identified as Acinetobacter pittii. Compared with growth in nutrient broth supplemented with OTA (OTA-NB), strain AP19 grew faster in nutrient broth (NB), but the ability of the resulting cell lysates to remove OTA was weaker. After cultivation in NB, the cell lysate of strain AP19 was able to remove 100% of 1 mg/L OTA within 18 h. The cell lysate fraction > 30 kDa degraded 100% of OTA within 12 h, while the fractions < 30 kDa were practically unable to degrade OTA. Further anion exchange chromatography of the > 30 kDa fraction yielded two peaks exhibiting significant OTA degradation activity. The degradation product was identified as OTα. Amino acid metabolism exhibited major transcriptional trends in the response of AP19 to OTA. The dacC gene encoding carboxypeptidase was identified as one of the contributors to OTA degradation. Soil samples inoculated with strain AP19 showed significant OTA degradation. These results provide significant insights into the discovery of novel functions in A. pittii, as well as its potential as an OTA decomposer.

Development of a growth-coupled selection platform for directed evolution of heme biosynthetic enzymes in Corynebacterium glutamicum.

Heme is an important tetrapyrrole compound, and has been widely applied in food and medicine industries. Although microbial production of heme has been developed with metabolic engineering strategies during the past 20 years, the production levels are relatively low due to the multistep enzymatic processes and complicated regulatory mechanisms of microbes. Previous studies mainly adopted the strategies of strengthening precursor supply and product transportation to engineer microbes for improving heme biosynthesis. Few studies focused on the engineering and screening of efficient enzymes involved in heme biosynthesis. Herein, a growth-coupled, high-throughput selection platform based on the detoxification of Zinc-protoporphyrin IX (an analogue of heme) was developed and applied to directed evolution of coproporphyrin ferrochelatase, catalyzing the insertion of metal ions into porphyrin ring to generate heme or other tetrapyrrole compounds. A mutant with 3.03-fold increase in k cat/K M was selected. Finally, growth-coupled directed evolution of another three key enzymes involved in heme biosynthesis was tested by using this selection platform. The growth-coupled selection platform developed here can be a simple and effective strategy for directed evolution of the enzymes involved in the biosynthesis of heme or other tetrapyrrole compounds.

Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde.

Methanol is a promising one-carbon feedstock for biomanufacturing, which can be sustainably produced from carbon dioxide and natural gas. However, the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide (NAD+)-dependent methanol dehydrogenase (Mdh) that oxidizes methanol to formaldehyde. Herein, the neutrophilic and mesophilic NAD+-dependent Mdh from Bacillus stearothermophilus DSM 2334 (MdhBs) was subjected to directed evolution for enhancing the catalytic activity. The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants. MdhBs variants with up to 6.5-fold higher Kcat/KM value for methanol were screened from random mutation libraries. The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity. The beneficial T153P mutation changes the interaction network of this residue and breaks the α-helix important for substrate binding into two short α-helices. Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve MdhBs, and this study provides an efficient strategy for directed evolution of Mdh.

Fairness-aware genetic-algorithm-based few-shot classification.

Artificial-intelligence-assisted decision-making is appearing increasingly more frequently in our daily lives; however, it has been shown that biased data can cause unfairness in decision-making. In light of this, computational techniques are needed to limit the inequities in algorithmic decision-making. In this letter, we present a framework to join fair feature selection and fair meta-learning to do few-shot classification, which contains three parts: (1) a pre-processing component acts as an intermediate bridge between fair genetic algorithm (FairGA) and fair few-shot (FairFS) to generate the feature pool; (2) the FairGA module considers the presence or absence of words as gene expression, and filters out key features by a fairness clustering genetic algorithm; (3) the FairFS part carries out the task of representation and fairness constraint classification. Meanwhile, we propose a combinatorial loss function to cope with fairness constraints and hard samples. Experiments show that the proposed method achieves strong competitive performance on three public benchmarks.

PFK2/FBPase-2 is a potential target for metabolic engineering in the filamentous fungus Myceliophthora thermophila.

The key enzyme 6-phosphofructo-2-kinase (PFK2)/fructose-2,6-bisphosphatase (FBPase-2) is responsible for regulating the rates of glycolysis and gluconeogenesis in eukaryotes. However, its functions and mechanisms in filamentous fungi remain largely enigmatic. In this study, we systematically investigated the function of this enzyme in Myceliophthora thermophila, a thermophilic filamentous fungus with great capacity to produce industrial enzymes and organic acids. Our results showed that the M. thermophila genome encodes three isomers, all with the PFK2/FBPase-2 structure: pfk2-a, pfk2-b, and pfk2-c. Overexpression of each gene revealed that endogenous expression of pfk2-c (PFK2 activity) promoted glucose metabolism, while overexpression of pfk2-a (FBPase-2 activity) inhibited strain growth. Using knockouts, we found that each gene was individually non-essential, but the triple knockout led to significantly slower growth compared with the wild-type strain. Only the pfk2-a single knockout exhibited 22.15% faster sugar metabolism, exerted through activation of 6-phosphofructo-1-kinase (PFK1), thereby significantly promoting glycolysis and the tricarboxylic acid cycle. The FBPase-2 deletion mutant strain also exhibited overflow metabolism, and knocking out pfk2-a was proved to be able to improve the production and synthesis rate of various metabolites, such as glycerol and malate. This is the first study to systematically investigate the function of PFK2/FBPase-2 in a thermophilic fungus, providing an effective target for metabolic engineering in filamentous fungi.

LaeA regulates morphological development and ochratoxin A biosynthesis in Aspergillus niger.

The global regulator LaeA and its orthologs govern the morphogenetic development and secondary metabolism of several filamentous ascomycetes. In Aspergillus niger, it has been shown that an LaeA ortholog (AnLaeA) regulates the production of citric acid and secondary metabolites. In this work, we constructed AnlaeA disruption and overexpression strains to investigate the roles of AnLaeA in morphological development and ochratoxin A (OTA) biosynthesis in A. niger. Phenotypic observation, chemical analysis, and gene expression analysis indicated that AnLaeA acts as a negative regulator of conidial morphogenesis and positively regulates gene expression of the OTA cluster in A. niger grown in CYA medium. However, it was observed that the upregulation of gene expression of the OTA cluster does not necessarily increase OTA production. Our results contribute to a better understanding of the AnlaeA regulatory mechanism and suggest the AnlaeA gene as a potential target for developing control strategies for A. niger infection and OTA biosynthesis.

Few-Shot Text Classification with Global-Local Feature Information.

Meta-learning frameworks have been proposed to generalize machine learning models for domain adaptation without sufficient label data in computer vision. However, text classification with meta-learning is less investigated. In this paper, we propose SumFS to find global top-ranked sentences by extractive summary and improve the local vocabulary category features. The SumFS consists of three modules: (1) an unsupervised text summarizer that removes redundant information; (2) a weighting generator that associates feature words with attention scores to weight the lexical representations of words; (3) a regular meta-learning framework that trains with limited labeled data using a ridge regression classifier. In addition, a marine news dataset was established with limited label data. The performance of the algorithm was tested on THUCnews, Fudan, and marine news datasets. Experiments show that the SumFS can maintain or even improve accuracy while reducing input features. Moreover, the training time of each epoch is reduced by more than 50%.

Deletion and Overexpression of the AnOTAbzip Gene, a Positive Regulator of Ochratoxin A Biosynthesis in Aspergillus niger.

The ochratoxin A (OTA) biosynthetic gene cluster includes a bZIP transcription factor (TF) gene (OTAbzip) that has been identified in different fungal species. However, most previous studies identified the OTAbzip gene in ochratoxigenic fungi using bioinformatics methods, while few studies focused on deleting the gene, let alone overexpressing it, to characterize the function of the OTAbZIP TF. Here, we characterized the AnOTAbZIP TF in an ochratoxigenic isolate of Aspergillus niger by deleting and overexpressing the AnOTAbzip gene and examining the role of AnOTAbZIP in morphological development, OTA biosynthesis, and pathogenicity. Chemical and gene expression analyses revealed that AnOTAbZIP positively regulates OTA biosynthesis, since the loss of OTA production and the downregulation of the OTA biosynthetic genes were observed in the ΔAnOTAbzip strain, compared with the wild-type (WT) and OE::AnOTAbzip strains. In terms of pathogenicity, the ΔAnOTAbzip strain produced a greater lesion on grape berries, especially with respect to the OE::AnOTAbzip strain, rather than WT. Finally, the ΔAnOTAbzip strain was also more tolerant to oxidative stress with respect to the OE::AnOTAbzip and WT strains in that order. These new findings improve our understanding of the AnOTAbZIP regulatory mechanism and help develop strategies to attenuate plant pathogenicity and reduce OTA biosynthesis of A. niger.

Deacetoxycephalosporin C synthase (expandase): Research progress and application potential.

Cephalosporins play an indispensable role against bacterial infections. Deacetyloxycephalosporin C synthase (DAOCS), also called expandase, is a key enzyme in cephalosporin biosynthesis that epoxides penicillin to form the hexavalent thiazide ring of cephalosporin. DAOCS in fungus Acremonium chrysogenum was identified as a bifunctional enzyme with both ring expansion and hydroxylation, whereas two separate enzymes in bacteria catalyze these two reactions. In this review, we briefly summarize its source and function, improvement of the conversion rate of penicillin to deacetyloxycephalosporin C through enzyme modification, crystallography features, the prediction of the active site, and application perspective.

Improving citric acid production of an industrial Aspergillus niger CGMCC 10142: identification and overexpression of a high-affinity glucose transporter with different promoters.

BACKGROUND: Glucose transporters play an important role in the fermentation of citric acid. In this study, a high-affinity glucose transporter (HGT1) was identified and overexpressed in the industrial strain A. niger CGMCC 10142. HGT1-overexpressing strains using the PglaA and Paox1 promoters were constructed to verify the glucose transporter functions. RESULT: As hypothesized, the HGT1-overexpressing strains showed higher citric acid production and lower residual sugar contents. The best-performing strain A. niger 20-15 exhibited a reduction of the total sugar content and residual reducing sugars by 16.5 and 44.7%, while the final citric acid production was significantly increased to 174.1 g/L, representing a 7.3% increase compared to A. niger CGMCC 10142. Measurement of the mRNA expression levels of relevant genes at different time-points during the fermentation indicated that in addition to HGT1, citrate synthase and glucokinase were also expressed at higher levels in the overexpression strains. CONCLUSION: The results indicate that HGT1 overexpression resolved the metabolic bottleneck caused by insufficient sugar transport and thereby improved the sugar utilization rate. This study demonstrates the usefulness of the high-affinity glucose transporter HGT1 for improving the citric acid fermentation process of Aspergillus niger CGMCC 10142.

Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization.

Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for the cost-effective production of biofuels and commodity chemicals. Galactose is one of the most abundant sugar in many hemicelluloses, and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fungus Myceliophthora thermophila has been used as a cell factory to produce biochemicals directly from renewable polysaccharides. In this study, we draw out the two native galactose utilization pathways, including the Leloir pathway and oxido-reductive pathway, and identify the significance and contribution of them, through transcriptional profiling analysis of M. thermophila and its mutants on galactose. We find that galactokinase was necessary for galactose transporter expression, and disruption of galK resulted in decreased galactose utilization. Through metabolic engineering, both galactokinase deletion and galactose transporter overexpression can activate internal the oxido-reductive pathway and improve the consumption rate of galactose. Finally, the heterologous galactose-degradation pathway, De Ley-Doudoroff (DLD) pathway, was successfully integrated into M. thermophila, and the consumption rate of galactose in the engineered strain was increased by 57%. Our study focuses on metabolic engineering for accelerating galactose utilization in a thermophilic fungus that will be beneficial for the rational design of fungal strains to produce biofuels and biochemicals from a variety of feedstocks with abundant galactose.

Enhancement of Acid Protease Activity of Aspergillus oryzae Using Atmospheric and Room Temperature Plasma.

Atmospheric and room temperature plasma (ARTP) system is a novel and efficient mutagenesis protocol for microbial breeding. In this study, ARTP was employed to treat spores of Aspergillus oryzae strain 3.042 for selection of high acid protease producers. With an irradiation time of 150 s at the lethal rate of 90%, 19 mutants with higher acid protease activity were initially selected based on different mutant colony morphology and ratio of the clarification halo of protease activity to the colony diameter. Measurements of the acid protease activity revealed that mutant strain B-2 is characterized by a steady hereditary stability with increased acid protease, neutral protease and total protease activities of 54.7, 17.3, and 8.5%, respectively, and decreased alkaline protease activity of 8.1%. In summary, the identified mutant strain B-2 exhibits great potential for the enhancement of the insufficient acid protease activity during the middle and later stages of soy sauce fermentation.

Disruption or reduced expression of the orotidine-5'-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger.

BACKGROUND: Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited. RESULTS: In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5'-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors. CONCLUSIONS: In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.

veA Gene Acts as a Positive Regulator of Conidia Production, Ochratoxin A Biosynthesis, and Oxidative Stress Tolerance in Aspergillus niger.

The veA gene is a key regulator governing morphogenetic development and secondary metabolism in many fungi. Here, we characterized and disrupted a veA orthologue in an ochratoxigenic Aspergillus niger strain. Morphological development, ochratoxin A (OTA) biosynthesis, and oxidative stress tolerance in the wild-type and veA disruption strains were further analyzed. Accordingly, the link between the veA gene and development of specific gene brlA, OTA biosynthesis key gene pks, and oxidative-stress-tolerance-related gene cat was explored. Results demonstrated that the veA gene acts as a positive regulator of conidia production, OTA biosynthesis, and oxidative stress tolerance in A. niger, regardless of light conditions. Darkness promoted conidial production and OTA biosynthesis in the A. niger wild-type strain. Our results contribute to a better understanding of the veA regulatory mechanism and suggest the veA gene as a potential target for developing control strategies for A. niger infection and OTA biosynthesis.

Functional analysis of the mitochondrial alternative oxidase gene (aox1) from Aspergillus niger CGMCC 10142 and its effects on citric acid production.

In this work, we constructed the aox1 disruption strains 3-4 and 4-10, as well as the aox1 overexpression strains 72 and 102 in Aspergillus niger. The energy metabolism, EMP, TCA pathways, and flux were investigated for the citric acid (CA) overproduction via the aox1 overexpression among them. As expected, the overexpression of the aox1 gene enabled a higher growth rate than that of the rate of its parent strain in medium with respiratory chain inhibitors. In liquefied corn starch medium supplemented with 0.2 µg/mL antimycin A, the CA production of the overexpression strain 102 reached up to 169.1 g/L, whereas the highest value of the parent strain was 158.9 g/L. For the perspective of the aox1 disruption strain 4-10, the yield of CA dropped to 125.6 g/L, and the loose mycelial pellets forming in the medium also revealed that the fundamentally important role of AOX in A. niger lies in the resistance to oxidative stress under fully aerobic conditions. Based on real-time qPCR gene expression analysis and measurement of intracellular ATP and NADH levels, we came to a conclusion that the higher NADH oxidation rate resulting from the overexpression of the aox1 gene mainly contributed to rate-limited step's acceleration and strengthened metabolic flow via mycelia and led to the CA yield in these strains increased by 13.5 and 10.8%, respectively. Subsequently, it was found that overexpression strains had higher AOX relative content and more oxygen consumption at different fermentation stages, which fully confirmed the close relationship between aox1 gene and energy metabolism, and comprehensively revealed aox1 gene function through the combination with the above conclusions.

Isolation and identification of histamine-producing Enterobacteriaceae from Qu fermentation starter for Chinese rice wine brewing.

Histamine (HIS) producers in fermented wines are generally believed to be lactic acid bacteria (LAB), and other microorganisms have received little or no attention. In this work, HIS-producing bacteria were isolated from Qu fermentation starter for Chinese rice wine brewing by decarboxylase medium, and their identity was confirmed by RP-HPLC and PCR. Surprisingly, the histidine decarboxylase gene (hdc) was present in only 2 out of 26 isolates. All 26 isolates were genotyped using the randomly amplified polymorphic DNA (RAPD)-PCR assay, which revealed the presence of 21 biotypes. Single type isolates were identified via 16S rRNA sequence analysis, in some cases coupled with partial sequencing of the rpoB or dnaJ gene. All isolates belonged to the Enterobacteriaceae, and included Enterobacter asburiae, Enterobacter cloacae, Enterobacter hormaechei, Citrobacter amalonaticus and Cronobacter sakazakii. All these strains were capable of producing >3.5 mg/L of HIS in TS medium without ethanol, but did not grow in TS medium with 8% ethanol. Small-scale Chinese rice wine fermentation revealed that HIS contents exhibited the same trend as the LAB and ethanol no matter what kinds of Qu were used. However, in the early stages of fermentation (from day 2 to day 4), the HIS contents had a stronger correlation with Enterobacteriaceae (0.943) than with LAB (0.369) when the Qu fermented samples are analyzed as a whole. Moreover, the lowest HIS content was measured in Xiao Qu (Q) fermented sample at the end of fermentation, which suggests that the formation of HIS in the early stages of fermentation has a decisive effect on HIS content in the final product. Our results demonstrate that Enterobacteriaceae from Qu are an important cause for HIS formation in Chinese rice wine. Consequently, selecting Qu with a low content of Enterobacteriaceae contaminants and inhibiting the growth of Enterobacteriaceae in the early stages of fermentation are useful approaches for preventing excessive amounts of HIS formation in Chinese rice wine brewing.

Purification and characterization of glutamate decarboxylase from Enterococcus raffinosus TCCC11660.

Glutamate decarboxylase (GAD) is the sole enzyme that synthesizes γ-aminobutyric acid through the irreversible decarboxylation of L-glutamate. In this study, the purification and characterization of an unreported GAD from a novel strain of Enterococcus raffinosus TCCC11660 were investigated. The native GAD from E. raffinosus TCCC11660 was purified 32.3-fold with a recovery rate of 8.3%, using ultrafiltration and ammonium sulfate precipitation, followed by ion-exchange and size-exclusion chromatography. The apparent molecular weight of purified GAD, as determined by SDS-PAGE and size-exclusion chromatography was 55 and 110 kDa, respectively, suggesting that GAD exists as a dimer of identical subunits in solution. In the best sodium citrate buffer, metal ions of Mo6+ and Mg2+ had positive effects, while Cu2+, Fe2+, Zn2+ and Co2+ showed significant adverse effects on enzyme activity. The optimum pH and temperature of GAD were determined to be 4.6 and 45 °C, while the K m and V max values for the sole L-glutamate substrate were 5.26 and 3.45 µmol L-1 min-1, respectively.

Comparative metabolomics reveals the mechanism of avermectin production enhancement by S-adenosylmethionine.

It was found that S-adenosylmethionine (SAM) could effectively improve avermectin titer with 30-60 µg/mL addition to FH medium. To clearly elucidate the mechanism of SAM on intracellular metabolites of Streptomyces avermitilis, a GC-MS-based comparative metabolomics approach was carried out. First, 230 intracellular metabolites were identified and 14 of them remarkably influenced avermectin biosynthesis were discriminative biomarkers between non-SAM groups and SAM-treated groups by principal components analysis (PCA) and partial least squares (PLS). Based on further key metabolic pathway analyses, these biomarkers, such as glucose, oxaloacetic acid, fatty acids (in soybean oil), threonine, valine, and leucine, were identified as potentially beneficial precursors and added in medium. Compared with single-precursor feeding, the combined feeding of the precursors and SAM markedly increased the avermectin titer. The co-feeding approach not only directly verified our hypothesis on the mechanism of SAM by comparative metabolomics, but also provided a novel strategy to increase avermectin production.

A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved in the Biosynthesis of Ochratoxin A in Aspergillus niger.

The polyketide synthase gene An15g07920 was known in Aspergillus niger CBS 513.88 as putatively involved in the production of ochratoxin A (OTA). Genome resequencing analysis revealed that the gene An15g07920 is also present in the ochratoxin-producing A. niger strain 1062. Disruption of An15g07920 in A. niger 1062 removed its capacity to biosynthesize ochratoxin ß (OTß), ochratoxin α (OTα), and OTA. These results indicate that the polyketide synthase encoded by An15g07920 is a crucial player in the biosynthesis of OTA, in the pathway prior to the phenylalanine ligation step. The gene An15g07920 reached its maximum transcription level before OTA accumulation reached its highest level, confirming that gene transcription precedes OTA production. These findings will not only help explain the mechanism of OTA production in A. niger but also provide necessary information for the development of effective diagnostic, preventive, and control strategies to reduce the risk of OTA contamination in foods.